scholarly journals Proteophosphoglycans of Leishmania mexicana: Molecular cloning and characterization of the Leishmania mexicana ppg2 gene encoding the proteophosphoglycans aPPG and pPPG2 that are secreted by amastigotes and promastigotes

1999 ◽  
Vol 344 (3) ◽  
pp. 787-795 ◽  
Author(s):  
Ulrich GÖPFERT ◽  
Nathan GOEHRING ◽  
Christian KLEIN ◽  
Thomas ILG
Keyword(s):  
Mrna Level ◽  
Single Copy ◽  
Gene Replacement ◽  
Mammalian Host ◽  
Cdna Expression Library ◽  
Dna Repeats ◽  
The Novel ◽  
Leishmania Mexicana ◽  
Expression Library ◽  
Gene Encoding

Intracellular amastigotes of the pathogenic protozoon Leishmania mexicana secrete an extensively phosphoglycosylated proteophosphoglycan (aPPG) into the phagolysosome of mammalian host macrophages, that appears to fulfil important functions for the parasites. Promastigotes (the sandfly vector forms) of the same species secrete a proteophosphoglycan with identical protein backbone but exhibiting stage-specific phosphoglycosylation patterns [Klein, Göpfert, Goehring, Stierhof and Ilg (1999) Biochem. J. 344, 775-786]. In this study we report the cloning of the novel repeat-containing proteophosphoglycan gene ppg2 by antibody screening of a Leishmania mexicana amastigote cDNA expression library. ppg2 is equally expressed in promastigotes and amastigotes at the mRNA level. Targeted gene replacement of both alleles of the single copy gene ppg2 results in the loss of pPPG2 expression in promastigotes. Antisera against Escherichia coli-expressedppg2 recognize the deglycosylated forms of aPPG as well as pPPG2. These results confirm that ppg2 encodes the protein backbones of aPPG and pPPG2. An unusual finding is that ppg2 exhibits two stable allelic forms, ppg2a and ppg2b. Their main difference lies in the number of central 72 bp DNA repeats (7 versus 8). ppg2a and ppg2b encode polypeptide chains of 574 and 598 amino acids, respectively, that show no homology to known proteins. The novel 24 amino acid Ser-rich peptide repeats encoded by the 72 bp DNA repeats are targets for Ser phosphoglycosylation in Leishmania mexicana.

scholarly journals Identification and Molecular Characterization of a Gene Encoding a Protective Leishmania amazonensis Trp-Asp (WD) Protein

2004 ◽  
Vol 72 (4) ◽  
pp. 2194-2202 ◽  
Author(s):  
Kimberly Campbell ◽  
Vsevolod Popov ◽  
Lynn Soong
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Single Copy ◽  
Interleukin 10 ◽  
Leishmania Amazonensis ◽  
Cdna Expression Library ◽  
Protein Protein Interactions ◽  
Expression Library ◽  
Gene Encoding ◽  
Mucocutaneous Leishmaniasis

ABSTRACT Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a ∼3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several other Leishmania species. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4+ cells of L. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response of L. amazonensis.

Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus

Parasitology ◽  
1999 ◽  
Vol 119 (4) ◽  
pp. 405-412 ◽  
Author(s):  
P. J. SKUCE ◽  
D. L. REDMOND ◽  
S. LIDDELL ◽  
E. M. STEWART ◽  
G. F. J. NEWLANDS ◽  
...  
Keyword(s):  
Haemonchus Contortus ◽  
Southern Blot Analysis ◽  
Single Copy ◽  
Blood Feeding ◽  
Cdna Expression Library ◽  
Cysteine Proteinases ◽  
Rt Pcr ◽  
Expression Library ◽  
Developmentally Regulated

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a ‘proteinase-enriched’ Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT–PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

scholarly journals Cloning and Characterization of an Ehrlichia canis Gene Encoding a Protein Localized to the Morula Membrane

2003 ◽  
Vol 71 (4) ◽  
pp. 2218-2225 ◽  
Author(s):  
Ching-Hao Teng ◽  
Raghavan U. M. Palaniappan ◽  
Yung-Fu Chang
Keyword(s):  
Recombinant Protein ◽  
Blot Analysis ◽  
Polyclonal Antibodies ◽  
Transmembrane Protein ◽  
Single Copy ◽  
Ehrlichia Canis ◽  
Chromosomal Dna ◽  
Expression Library ◽  
Gene Encoding ◽  
Host Cytoplasm

ABSTRACT A gene encoding a 23.5-kDa ehrlichial morula membrane protein designated MmpA was cloned by screening an Ehrlichia canis expression library with convalescent dog sera, which resulted in three positive clones. Sequence analysis of the insert DNAs from all three clones indicated an open reading frame with a size of 666 bp that encodes MmpA. The structural analysis of MmpA indicated that it is a transmembrane protein with extreme hydrophobicity. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated the presence of a single copy of the mmpA gene in E. canis and Ehrlichia chaffeensis but not in the human granulocytic ehrlichiosis agent. The mmpA gene was amplified, cloned, and expressed as a fusion protein. Polyclonal antibodies to the recombinant protein (rMmpA) were raised in rabbits. Western blot analysis of E. canis and E. chaffeensis lysates with the anti-rMmpA serum resulted in the presence of an MmpA band only in E. canis, not in E. chaffeenesis. Sera from dogs which were either naturally or experimentally infected with E. canis recognized the recombinant protein. Double immunofluorescence confocal microscopy studies demonstrated that MmpA was localized mainly on the morula membrane of E. canis. Since the morula membrane is the interface between the ehrlichial growing environment and the host cytoplasm, MmpA may play a role in bacterium-host cell interactions.

scholarly journals Glycosylation Defects and Virulence Phenotypes ofLeishmania mexicana Phosphomannomutase and Dolicholphosphate-Mannose Synthase Gene Deletion Mutants

2001 ◽  
Vol 21 (23) ◽  
pp. 8168-8183 ◽  
Author(s):  
Attila Garami ◽  
Angela Mehlert ◽  
Thomas Ilg
Keyword(s):  
Gene Deletion ◽  
Open Reading Frames ◽  
Mammalian Host ◽  
Deletion Mutants ◽  
Leishmania Mexicana ◽  
Gpi Anchor ◽  
Gene Encoding ◽  
Mouse Macrophages ◽  
Ample Supply ◽  
Reading Frames

ABSTRACT Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (ΔPMM and ΔDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicanaΔDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana ΔPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.

scholarly journals The major parasite surface antigen associated with human resistance to schistosomiasis is a 37-kD glyceraldehyde-3P-dehydrogenase.

1989 ◽  
Vol 170 (6) ◽  
pp. 2065-2080 ◽  
Author(s):  
V Goudot-Crozel ◽  
D Caillol ◽  
M Djabali ◽  
A J Dessein
Keyword(s):  
Recombinant Protein ◽  
Surface Antigen ◽  
Single Copy ◽  
Glycolytic Enzyme ◽  
Human Populations ◽  
Cdna Expression Library ◽  
T Cell Epitopes ◽  
Expression Library ◽  
Major Health Problem ◽  
Susceptibility To Infection

Schistosomiasis, due to Schistosoma mansoni, is a major health problem in many subtropical countries, and major efforts are being made to define a vaccine. In this regard, we have reported that sera from subjects with low susceptibility to infection by S. mansoni react with a major larval surface antigen (P-37), having an apparent molecular mass of 37 kD, against which sera of susceptible individuals show little reactivity. We have now cloned the cDNA for this antigen by screening a schistosome cDNA expression library with antibodies against the purified protein. The selected cDNAs encode a protein that is specifically identified by immune human sera containing antibodies against P-37, while sera exhibiting low or no reactivity toward P-37 fail to recognize the recombinant protein. The cloned cDNAs hybridize with a 1.2-kb RNA that is the transcript of a single copy gene. This RNA directs the synthesis of a 36.5-kD polypeptide that is precipitated by sera from the most resistant subjects. The amino acid sequence of the encoded polypeptide shows homology with the glycolytic enzyme Glyceraldehyde-3P-dehydrogenase (72.5% of positional identity with human Glyceraldehyde-3P-dehydrogenase). Antibodies against the recombinant protein identified P-37 on the larva. These findings, together with other reports, indicate that a number of conserved proteins may be major targets of host-protective immunity against S. mansoni. The hypothesis is discussed that genetic restriction of the immune response to these antigens may occur in heterogeneous human populations because of the limited number of T cell epitopes carried by these host-like proteins. Such genetic effects might allow parasite transmission through nonresponder (susceptible) individuals. This hypothesis and the protective properties of P-37 can now be tested using the recombinant protein and synthetic peptides derived from selected regions of the polypeptide chain.

scholarly journals Molecular characterization of a novel transcription factor that controls stromelysin expression.

1995 ◽  
Vol 15 (6) ◽  
pp. 3164-3170 ◽  
Author(s):  
L Sanz ◽  
J Moscat ◽  
M T Diaz-Meco
Keyword(s):  
Transcription Factor ◽  
Leucine Zipper ◽  
Critical Role ◽  
Single Copy ◽  
Binding Activity ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Binding Domains ◽  
3T3 Fibroblasts ◽  
Simplex Virus

Stromelysins, which are the metalloproteinases with the widest substrate specificities, play a critical role in tumor invasion and metastasis. We have previously reported an element (SPRE) of the stromelysin promoter located between nucleotides -1221 and -1203 that is necessary and sufficient for the control of stromelysin gene expression by mitogenic activation, which induces a nuclear activity that binds to this sequence. Using a concatenated probe with several copies of this element to screen a lambda gt11 cDNA expression library from mouse Swiss 3T3 fibroblasts, we report here the molecular cloning of a cDNA coding for a novel protein (SPBP) of 937 amino acids that binds to this element and has several features of a transcription factor, such as a putative leucine zipper region, a nuclear localization signal, and a basic domain with homology to the DNA-binding domains of Fos and Jun. Evidence that SPBP is at least a critical component of the mitogen-induced SPRE nuclear binding activity is presented here. Furthermore, the transfection of an expression plasmid for SPBP transactivates reporter chloramphenicol acetyltransferase plasmids containing either the full-length stromelysin promoter or a single copy of the SPRE cloned upstream of the herpes simplex virus thymidine kinase minimal promoter. Therefore, the results presented here identify a novel transcription factor critically involved in the control of stromelysin expression.

scholarly journals Mutation of RNA Polymerase β Subunit (rpoB) Promotes hVISA-to-VISA Phenotypic Conversion of Strain Mu3

2011 ◽  
Vol 55 (9) ◽  
pp. 4188-4195 ◽  
Author(s):  
Miki Matsuo ◽  
Tomomi Hishinuma ◽  
Yuki Katayama ◽  
Longzhu Cui ◽  
Maria Kapi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Response Regulator ◽  
Single Copy ◽  
Gene Replacement ◽  
Vancomycin Resistance ◽  
Β Subunit ◽  
Gene Encoding ◽  
Content Type ◽  
Replacement Method ◽  
Two Component

ABSTRACTThe clinical vancomycin-intermediateStaphylococcus aureus(VISA) strain Mu50 carries two mutations in thevraSRandgraRStwo-component regulatory systems (TCRSs), namely,vraS(I5N) andgraR(N197S) (hereinafter designatedgraR*). The clinical heterogeneously vancomycin-intermediateS. aureus(hVISA) strain Mu3 shares with Mu50 the mutation invraSthat encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR*, carrying the mutated two-component response regulatorgraR*, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy ofgraR* into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR* thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50rpoBgene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR* with rifampin gave rise torpoBmutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR* were found to possessrpoBmutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneousrpoBmutants with various degrees of increased vancomycin resistance.

scholarly journals Description of a novel eukaryotic deoxyuridine 5′-triphosphate nucleotidohydrolase in Leishmania major

1997 ◽  
Vol 325 (2) ◽  
pp. 441-447 ◽  
Author(s):  
Ana CAMACHO ◽  
Rosalía ARREBOLA ◽  
Javier PEÑA-DIAZ ◽  
Luis M. RUIZ-PÉREZ ◽  
Dolores GONZÁLEZ-PACANOWSKA
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Leishmania Major ◽  
Active Form ◽  
Single Copy ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Highly Active

A Leishmaniamajor full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichiacoli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichiacoli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.

Disruption of TRI101, the Gene Encoding Trichothecene 3-O-Acetyltransferase, fromFusarium sporotrichioides

1999 ◽  
Vol 65 (12) ◽  
pp. 5252-5256 ◽  
Author(s):  
Susan P. McCormick ◽  
Nancy J. Alexander ◽  
Susan E. Trapp ◽  
Thomas M. Hohn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusarium Graminearum ◽  
Parent Strain ◽  
Cdna Expression Library ◽  
Fusarium Sporotrichioides ◽  
Expression Library ◽  
Gene Encoding ◽  
Cdna Expression ◽  
Yeast Transformants

ABSTRACT We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is theF. sporotrichioides homolog of the Fusarium graminearum TRI101 gene. Mutants of F. sporotrichioides NRRL 3299 produced by disruption ofTRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3,15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods

Parasitology ◽  
2010 ◽  
Vol 138 (3) ◽  
pp. 333-343 ◽  
Author(s):  
D. S. ZARLENGA ◽  
A. J. NISBET ◽  
L. C. GASBARRE ◽  
W. M. GARRETT
Keyword(s):  
Signal Sequence ◽  
Blood Feeding ◽  
Cellular Level ◽  
Extracellular Nucleotides ◽  
Cdna Expression Library ◽  
The Novel ◽  
Native Protein ◽  
Expression Library ◽  
Bed Bug ◽  
Teladorsagia Circumcincta

SUMMARYApyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

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